The efficient transmission of influenza A virus (IAV) to new hosts is dependent on multiple factors. Cleavage-activation and membrane fusion characteristics are significant but overlooked properties of the IAV hemagglutinin (HA) protein that may influence transmission. The efficiency of HA cleavage-activation by proteases encountered in natural environments may impact virus infectivity, stability, and pathogenicity, and the pH at which HA-mediated fusion occurs may have broad implications for the stability and transmissibility of IAVs in nature. IAVs are exposed to highly varied selective pressures during natural and cross-species transmission cycles, which might necessitate the evolution of HA proteins having different stability phenotypes. Of the 16 delineated HA subtypes, the only HA subtypes to become established in the human population are H1, H2, and H3, as derived from the four major pandemics within the last century: H1N1 (1918), H2N2 (1957), H3N2 (1968), and H1N1 (2009). Although viruses having H5 and H7 HA subtypes have been shown to infect with high mortality rates, they have yet to gain a foothold in the human population due to inefficient intra-species transmission. Analysis of the available crystal structures of different HA subtypes have revealed marked structural variations. These structural differences are the basis for the classification of HA subtypes into two groups: group 1 and group 2. The aim of the current application is to examine phenotypic differences between the 16 HA subtypes by examining the role of group-specific residues in mediating the acid-induced destabilization of the meta-stable HA during its transition to a stable, fusogenic structure. The studies outlined in this application will provide the first large-scale, detailed evaluation of the protease cleavage-activation and membrane fusion properties for all 16 HA subtypes. In addition, the phenotypic effects of variations in the pH at which HA-mediated membrane fusion occurs will be examined with regard to the environmental persistence and stability of IAVs having acid-stable versus acid-labile HA proteins, as well as the efficiency of transmission in avian and mammalian species. Understanding the phenotypic characteristics of HA proteins representative of all 16 subtypes, as well as the role of both naturally occurring and group specific sequence variations, is essential if we are to derive information regarding the ease of cross-species transmission and the emergence of novel viruses in humans. PUBLIC HEALTH RELEVANCE: Influenza virus is a major public health threat to all sectors of the population that yields serious and deleterious social and economic consequences. Understanding the molecular factors that alter the efficiency of influenza infection in animal and human hosts is essential to understanding how new viruses may affect humans.